Enzymes of the .beta.-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase

Primary and secondary kinetic and secondary equilibrium deuterium isotope effect studies on the cis,cis-muconate cycloisomerase catalyzed interconversion of cis,-cis-muconate (CCM) and (+)-muconolactone (ML) have been performed. The primary and solvent kinetic deuterium isotope effects upon Vmax for the reactions of (+)-[5R-2H]muconolactone in water (HOH) and (+)-muconolactone in deuterium oxide (DOD) to form cis,cis-muconate are about 2.5-2.6 with the heavier isotopic reactions being the slower ones. The secondary equilibrium isotope effect for the formation cis,-cis-[2,3,4,5-2H4] muconate from (+)-[2,3,4,5S-2H4]muconolactone is 1.32 for KH/KD = [( cis,cis-muconate]/[(+)-muconolactone])/ [(+)-[2,3,4,5S-2H4]muconolactone]) and agrees well with the measured value of 1.45 on the basis of the fumarase reaction [Cook, P. F., Blanchard, J. S., & Cleland, W. W. (1980) Biochemistry 19, 4853-4858]. The secondary kinetic deuterium isotope effect determined by the equilibrium perturbation method [Cleland, W. W. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., & Northrop, D. B., Eds.) pp 153-175, University Park Press, Baltimore, MD] for the conversion of cis,cis-[2,3,4,5-2H4] muconate to (+)-[2,3,4,5S-2H4]muconolactone is 0.66, expressed as (VmaxCCM(H)/KmCCM(H]/(VmaxCCM(D)/KmCCM(D]. From the equilibrium and kinetic secondary deuterium isotope effects, the calculated value for the kinetic secondary deuterium isotope effect for the reverse reaction, (VmaxML(H)/KmML(H]/(VmaxML(D)/KmML(D], is about 0.96.